This program is comprised of four highly interactive projects from Baylor College of Medicine (BCM) and M.D. Anderson Cancer Center (MDACC) in the Texas Medical Center (TMC) in Houston that focus on different aspects of the regulation of self-renewal and differentiation in human embryonic stem (hES) cells. The projects examine the role of and interactions between four proteins that have been shown to play a functional role in ES cells, namely nanog, Germ Cell Nuclear Factor (GCNF), the tumor suppressor protein p53, and a novel protein, THAP11. Our overall goal is to use this collaborative program project mechanism to understand how these proteins and the complexes with which they associate interact to regulate hES. Our program investigators have unique strengths in chromatin regulation, protein-protein interaction, gene repression, and hES biology that, when combined, will enable us to make a major contribution to the understanding of the regulation of pluripotency and differentiation in hES. Project 1 (Z. Songyang) involves study of proteins that interact with NANOG, and the mechanism through which NANOG represses genes involved in differentiation, particularly with regard to epigenetic regulation through the polycomb PRC1 complex. Project 2 (A. Cooney) is focused on the role of GCNF in repression of pluripotency genes, which is required for differentiation. Project 3 (M. Barton) focuses on p53, which has been shown to repress nanog in mouse ES cells. In Project 4 (T. Zwaka), THAP11 functions in hES will be examined. THAP11 has multiple features which potentially link epigenetic regulation of DNA and histones;THAP11 also can regulate suz12, a component of the PRC2 complex required for PRC1 activity, thus linking THAP11 with NANOG. The relevance of all these protein interactions for hES pluripotency and differentiation will be tested in multiple collaborative experiments. The projects will be supported by three cores. An Administrative Core will provide centralized support for all projects, and will also sponsor three pilot projects that will be competitively selected annually, enabling us to expand the number of investigators in the TMC who work with hES. We will sponsor a hES Training and Culture Core that will train TMC investigators in hES technologies in small bimonthly workshops. This core will also support the Projects and Pilot Project in hES maintenance, quality control and large-scale expansion. A Genetic Modification Core will offer tailored development of knock-in, knock-out, and tetracycline-regulated gene expression in hES cells. The overall program involves investigators from the University of Texas-Southwestern in Dallas as well as from BCM and MDACC. By bringing hES training and pilot grant funds into this region of the Southwest, our program will make hES technology accessible to a very large group of investigators and substantially strengthen hES expertise regionally.